Lysosomal protein targeting sequence and therapeutic applications of same

ABSTRACT

The present disclosure relates to methods of treating diseases states such as lysosomal storage diseases and/or neurodegenerative diseases. Also disclosed are one or more compositions that may be useful for one or more of the disclosed methods, including compositions that may comprise acid β-glucosidase (GCase) protein comprising one or more mutations, peptides of acid β-glucosidase, and DNA vectors and cell lines related to acid β-glucosidase peptides or proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication 61/882,272, filed Sep. 25, 2013, which is incorporated inits entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DK036729 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

For integral lysosomal membrane proteins, specific peptide sequences areessential for their delivery to the lysosomes. These are theM6P-independent targeting systems that rely on peptide sequences and notM6P or other carbohydrate recognition systems. Inherent in the originalconcept of the lysosome, as described by deDuve (1), is an essentialmechanism to localize or target specific proteins/enzymes to thissubcellular organelle. Goldstein and Brown were awarded a Nobel Prizefor their discovery and elucidation of specific receptors on cells forthe uptake of extracellular low-density lipoproteins into the cell anddelivery to the lysosomes (2, 3). Later this concept ofreceptor-mediated endocytosis was generalized to include not onlyextracellular ligand-receptor interactions, but also the essential needfor such a system within cells for delivery/targeting of newlysynthesized proteins to the lysosomes. One such system was elucidated byKornfeld and coworkers (4) and Sly and coworkers (5, 6) and is now knownas the mannose-6-phosphate (M6P) system. This M6P is used by many of thesoluble proteins for their delivery to the lysosomes. The deficiency ofthe ability to attach M6P to such proteins results in the secretion ofseveral dozen proteins from the cell and a progressive multisystem fataldisease termed MLII/MLIII (7).

M6P and mannose receptor systems form the essential basis for thedelivery of administered recombinant proteins for the treatment of theso-called lysosomal storage diseases (8). Production of lysosomalproteins for delivery to specific affected tissues is completelydependent upon understanding what receptor is critical to those tissuesfor uptake, internalization, and delivery of the therapeutic proteins tothe lysosomes (8). For example, the mannose-receptor is significantlyrepresented on macrophages and other cells of the reticuloendothelialsystem (RES). Gaucher disease has its major disease manifestations inmacrophages and the RES. Effective enzyme therapy was developed based onthe ability to create recombinant acid β-glucosidase, the defectiveenzyme in Gaucher disease, with mannose terminated oligosaccharideresidues (9, 10). For Fabry disease, Pompe disease andMucopolysaccharidoses I, II, and VI, recombinant enzymes with M6Poligosaccharides have become the standards of care for the treatment ofthese diseases (e.g. 11, 12). The successes in treating these formerlyfatal and severely debilitating diseases have been documentedextensively. The total sales of enzymes for the treatment of thesediseases now exceed $5B annually.

There remain several membrane associated, not integral membrane,proteins that appear to have a completely different system for targetingto the lysosomes. One such protein is an enzyme, termed acidβ-glucosidase, which is defective in Gaucher disease, the most commonlysosomal storage disease (13). Acid β-glucosidase (also referred to asD-glucosyl-N-acylsphingosine glucohydrolase, glucocerebrosidase, GCase,assigned Enzyme Commission No. EC 3.2.1.45) is a lysosomalexoglycosidase for β-glucose-terminated sphingolipids (14,15).Insufficient activity of GCase is causal to the variants of Gaucherdisease, a common lysosomal storage disease (16). The human or mousegenes, GBA1 or Gba1, respectively, are about 7.5 kb and contain 11exons, which encodes a highly (˜86% identical/92% similar) conservedamino acid sequence. Over 300 mutations of various types have been foundin association with the variants of Gaucher disease and some have clearprognostic value for affected patients (17). Each of the resultantdifferent single amino acid substitutions lead to GCases with alteredcatalytic, stability, or both defects (e.g., 18). GCase is translatedfrom mRNAs into a protein that contains two functional, in tandem,leader sequences that differ in length, either 39(SEQ ID NO 24) or 19amino acids (19). The preferred initiation codon is not known.

Mature GCase is a glycoprotein of 497 amino acids (FIG. 3, SEQ ID NO 2)that is produced by co-translational glycosylation of four of fiveN-glycosylation sequences (N463 not occupied) of which only N-19 isessential for the formation of a catalytically active conformer (20).The enzyme also has properties of a membrane associated, but nottransmembrane, protein. Unlike most soluble lysosomal proteins that aretrafficked to the lysosome by the mannose-6-phosphate (M6P) receptorsystem (21,22), GCase contains little if any M6P (23). Newly synthesizedunglycosylated GCase is not secreted out of cells nor is enzyme secretedfrom I-Cell fibroblasts, which are deficient in the enzyme needed forM6P (21). Thus, the targeting to the lysosome of newly synthesized,intracellular, GCase is not oligosaccharide dependent (24).

Like the M6P targeting systems, non-carbohydrate-mediated lysosomaltargeting disruptions lead to multisystem fatal diseases (8, 9).Lysosomal Integral Membrane Protein 2 (LIMP-2) has been identified as atrafficking receptor for GCase (23, 25, 26). LIMP-2 is a 478 amino acid,85 Kd glycoprotein protein that is also known as SCARB-2/CD36L2. Thisprotein is present in the ER, Golgi, endosomal, lysosomal, and plasmamembrane compartments of cells (26, 27). As the name indicates, LIMP-2is an integral membrane protein with N- and COOH-terminal transmembranedomains, and a luminal domain (ldLIMP-2) that binds GCase (23) andpotentially other proteins. LIMP-2 binds to GCase in the ER (pH-6.8) andthe enzyme remains bound to LIMP-2 during its transport through theGolgi, trans-Golgi network, and endosomal compartments. LIMP-2 deliversGCase to the lysosomes after an acidic pH-modulated dissociation of thereceptor and ligand in the late endosomal compartment with liberation ofGCase. This dissociation is mediated by LIMP-2 histidine 171 (28). Noother proteins have been identified to bind to LIMP-2 inside of cells.

LIMP-2/SCARB-2 is also a scavenger receptor on the plasma membrane thatbinds peptide sequences of viruses, in particular enteroviruses (e.g.EV71), for internalization, lysosomal delivery and degradation (29-32).The ligand amino acid sequence of EV71 for human LIMP-2 has beenidentified within VP1 (30), which has no homology to GCase sequences.The corresponding receptor sequence on LIMP-2 is between amino acids144-151 (28). Other LIMP-2/SCARB-2 protein ligands that bind at theplasma membrane include KCNQ1, KCNE2, and Megalin (33).

Humans and mice with mutations in the LIMP-2 gene develop characteristicneurologic and renal diseases, but do not exhibit gross findings ofGaucher disease, i.e., GC storage or Gaucher cells (33, 34). The humandiseases associated with LIMP-2 mutations are termed the actionmyoclonus-renal failure syndrome (34). LIMP-2 deficient cells in humansand mice exhibit excess secretion of GCase out of the cells and intoplasma or culture media, but little GC accumulation in tissues (34, 23).LIMP-2 variants have also been implicated as potential modifiers in thedevelopment of Parkinson/Alzheimer diseases (35, 36, 33), as have GBA1mutations (36-39). Disruption of appropriate trafficking of GCase to thelysosome may provide a mechanistic basis for the impact of GBA1/Gba1mutations in the modification of α-synuclein metabolism and its role inParkinson disease (37, 38, 40). The impact of LIMP-2 on the expressionof Gaucher disease and both GCase and LIMP-2 variants as modifiers ofsynucleinopathies highlight the importance of understanding theinteractions of GCase and LIMP-2 and the localization of synthesizedGCase to the lysosome.

Thus, there remains a need for methods and compositions effective fortreating lysosomal storage diseases related to defects in the acidβ-glucosidase pathway such as Gaucher disease. Further, there is a needfor treatments of disease states related to dysregulation of synucleinmetabolism that may result from, or be exacerbated by, disruption ofappropriate trafficking of GCase to the lysosome, such asneurodegenerative disease states including Parkinson disease, Alzheimerdisease, and Lewy body disease. Finally, there remains a need forimproved methods of synthesizing recombinant GCase, which cansubsequently be used to treat disease states resulting from deficienciesin this enzyme. The instant disclosure seeks to address one or more ofthese needs.

BRIEF SUMMARY

Disclosed herein are methods of treating disease states includinglysosomal storage diseases and/or neurodegenerative diseases. Furtherdisclosed are compositions useful for the disclosed methods.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1: GCase-GFP transfection constructs for Gba1 null/null cells. Onthe left are the resultant GCases following the various deletions andindicated as GCaseZZZ to show the encoded mature amino acid contents ofthe expressed enzymes. On the constructs are the labels GCase-XX or XXXto designate the number of amino acids that were deleted from the —COOHend of the mature 497 amino acid sequence of the WT GCase. GFP wascloned in frame with the WT or GCase-XX or XXX. These constructs weresubsequently transfected into Gba1−/− fibroblasts.

FIG. 2: Co-localization of GCase-XX- or -XXX-GFP with lysotracker.Pearson Indices were obtained (see Methods) for co-localization of theWT or truncated GCases using lysotracker to label the lysosomes. Theconstruct containing only GFP (N-GFP) showed no co-localization.Compared to the WT sequence, GCase-23 and -75 showed only smalldecreases in lysosomal localization. GCase-150 and -225aa showeddecreases in co-localization with lysotracker to nearly background(N-GFP) levels. GCase-150aa and -225aa showed much more extensiveretention in the ER and Golgi compared to WT, GCase-23aa or GCase-75aa(data not shown).

FIG. 3: Mature WT GCase amino acid sequence highlighting the regionstargeted for mutagenesis, expression, and binding analyses. The aminoacid sequence of GCase is marked with the various GCase-XX or XXX inblue to indicate the deleted parts of the enzyme. Shown in green is atypical dileucine sequence for indirect targeting of membrane boundlysosomal proteins. Shown in red are the amino acids for targetedmutagenesis and binding studies to ldLIMP-2. FIG. 13 shows the highconservation of these sequences.

FIG. 4: Structure of WT GCase highlighting the potential ldLIMP2 bindingsequence localization. The two amino acid sequences from FIG. 3 weremapped to the crystal structure of WT human GCase. The orientation ofthe sequences is indicated by the amino acid numbers in the highlighted(Yellow or Orange) regions. The domains of GCase are shown in variouscolors as are the disulfides. The potential ldLIMP2 binding (Yellow,D399 to D409) sequence forms a surface accessible loop in domain I. Thissequence, DSPIIVDITKD (SEQ ID NO 3), and the DDQRLLL (SEQ ID NO 14)(Orange, D282 to L288 in Domain III) sequence are highly conserved inGCases from insects to humans. In red are the acid-base (E235) andnucleophile (E340) in catalysis, and N370S and L444P are commonmutations causal to Gaucher disease.

FIG. 5: Co-localization of GCases with specific alanine substitutions inthe TKD sequence. GCase-YFP constructs were transfected into Gba1−/−fibroblasts and co-localization was evaluated with either calnexin orcalreticulin (Green) for the ER/Golgi. The alanine (A) substitutions areas indicated in the WT TKD sequence. For the AAA and AKA GCases, themajority of the enzymes co-localized to the ER/Golgi (ER), whereas theAKD GCase showed about 50% of the enzyme localized to the lysosome(Lys).

FIG. 6: Immunoprecipitation of the alanine substituted GCases in the TKDsequence with ldLIMP2. In panels A to D, the lanes 1 through 6correspond to the band densities indicated by the bars in E. The panelsshow that the WT sequence (A) had nearly complete binding with ldLIMP2,i.e., retention on the beads to which ldLIMP2 was bound (Lane 4) and noGCase in the wash (Lane 5). In comparison, the triple alanine mutant (B)shows essentially no binding to ldLIMP2, i.e., all the GCase is in thewash (Lane 5). The AKA GCase mutant (C) showed about equal amounts ofGCase in Lanes 4 and 5 or about 50% binding to ldLIMP2. The AKD GCasemutant (D) had binding pattern that was similar to the WT sequence, butwith more GCase in the wash (Lane 5), i.e., somewhat less binding. Thequantitative results are shown in (E) in which 1, 2, 3, and 4 correspondto WT, AAA, AKA, and AKD, respectively.

FIG. 7: Binding and competition of fluorescent labeled or unlabeledDSPIIV (SEQ ID NO 15) GCase peptides to ldLIMP2. The change (δmP) influorescence polarization is plotted on the ordinate and the increasingconcentrations of the various peptides are on the abscissa. (A) Theconcentration of ldLIMP2 (50 nM) was fixed. With the WT (DSPIIV (SEQ IDNO 15)) peptide (duplicate experiments in  and ▾), saturation kineticswith about half maximal binding to ldLIMP2 was observed at 50 nM. TheDSPAIV (SEQ ID NO 17) and DSPIAV (SEQ ID NO 18) mutants did not showsaturation up to 250 nM, indicating their poor interaction with ldLIMP2.The ASPAAP peptide showed background changes. The ASPIIV peptide (SEQ IDNO 16) showed δmP values slightly above background indicating littlebinding to ldLIMP2. (B) To ensure that the label did notinterfere/promote binding, similar studies were conducted usingfluorescently labeled peptides as binders and their respective unlabeledpeptides as competitors. Each of the unlabeled peptides “competed” withthe labeled peptides in the expected ratios. (C) Labeled (WT*) andunlabeled (WT) peptides were used in complementary competition studies.Either the labeled or unlabeled WT peptides equally competed for bindingto ldLIMP2 showing both were equally effective in binding and that thelabel did not change the properties of the peptide-ldLIMP2 interaction.

FIG. 8: Immunoprecipitation of GCase in the presence of ldLIMP2 andincreasing molar ratios of the WT (DSPIIV (SEQ ID NO 15)) peptide. (A)Purified WT GCase was preincubated with purified ldLIMP2 in a molarratio of 1:1. Then, unlabeled DSPIIV (SEQ ID NO 15) was added in varyingmolar excesses (0 to 5×, top of figure) over GCase, incubated, and thenimmunoprecipitated with Protein G-coupled anti-LIMP-2 antibody beads.The beads were then eluted and the eluants were analyzed for GCase andldLIMP2 on Western blots using the specified antibodies. The resultsshow decreasing amounts of bound GCase with increasing peptide molarratios, i.e., peptide DSPIIV (SEQ ID NO 15) competed bound GCase off ofldLIMP2. In the bottom panel, ldLIMP2 recovery was the same at allpeptide ratios. (B) Purified ldLIMP2 was preincubated with unlabeled WTpeptide at various molar ratios. Then, purified WT GCase was added in a1:1 molar ratio with ldLIMP2, incubated, and then immunoprecipitated andprocessed as in (A). The results show that the peptide prevents thebinding of ldLIMP2 and WT GCase. (C) Purified WT GCase was preincubatedwith purified ldLIMP2 in a molar ratio of 1:1. Then peptide DDQRLLL, apotential candidate for the GCase ligand for ldLIMP2, was added invarying molar excesses over WT GCase, incubated, and thenimmunoprecipitated and processed as in (A). The results show thatpeptide DDQRLLL (SEQ ID NO 14) did not compete GCase off of ldLIMP2,thereby showing that this peptide did not have specificity for the GCasebinding site on ldLIMP2. DDQRLLL (SEQ ID NO 14) is a dileucine peptideoutside of the localized targeting region of DSPIIVDITKD (SEQ ID NO 3)(see FIG. 3).

FIG. 9: Effect pH on binding WT and alanine substituted DSPIIV (SEQ IDNO 15) peptides to ldLIMP2. The binding of the different peptides toldLIMP-2 showed little effect of alanine substitutions at pH-5.8. Incomparison, incremental decreases were evident in ldLIMP2 binding atpH=6.8, the approximate pH of the ER/cis-Golgi, as follows: the triplemutant (ASPAAV) having the lowest binding (<10% of WT), the DSPAIV (SEQID NO 17) (I402A) and DSPIAV (SEQ ID NO 18) (I403A) mutants beingintermediate (˜40-50% of WT), and ASPIIV (SEQ ID NO 16) (D399A) havingthe least change (˜50-60% of WT), relative to WT.

FIG. 10: Intracellular localization of GCase DSPIIV (SEQ ID NO 15)variants in Gba1−/− cells. (A) Typical examples of immunofluorescencelocalization of DSPIIV (SEQ ID NO 15) substituted GCase variantsfollowing transient transfections. The DSPIIV (SEQ ID NO 15) (WT) andESPIIV (SEQ ID NO 20) show co-localization of GCase (FITC) with eitherLamp1 (Red) or LIMP-2 (purple), indicating that the retention of chargeby E399 does not impact localization. The cells are typical for eitherD399 (DSPIIV (SEQ ID NO 15)) or E399 (ESPIIV (SEQ ID NO 20)). The DSPAIV(SEQ ID NO 17) (1402A) or DSPIAV (SEQ ID NO 18) (I403A) mutant shows noco-localization with ldLIMP2 or Lamp1, i.e. no binding to ldLIMP2 in thecell and no localization to the lysosome (Lamp1/ldLIMP2). The black andwhite figures in the lower right indicate the relative localization ofLAMP-1 (lysosomes) and WT GCase (ER/Golgi and Lysosomes) in Gba1−/−fibroblasts for comparison. (B) Pearson Indices for co-localization ofthe various mutants in the ER/Golgi (black) or lysosome (hatched). Thesubstitution of E for D (WT) at 399 (ESPIIV (SEQ ID NO 20)) did notalter the co-localization compared to WT. In comparison ASPIIV (SEQ IDNO 16) (D399A) and the double mutant, DSPAAV (SEQ ID NO 19)(1402A+I403A) showed little co-localization to the lysosome, butsignificant retention in the ER/Golgi. The single mutants, DSPAIV (SEQID NO 17) (1402A) and DSPIAV (SEQ ID NO 18) (I403A) showed partialco-localization to the lysosome, but greater retention in the ER/Golgi.

FIG. 11: Secretion and retention of GCase with alanine variants in theTDK (A) or DSPIIV (SEQ ID NO 15) (B and C) sequences. (A) Activities inthe media of Gba1−/− fibroblasts following transient transfections ofeach of the specified alanine substituted GCases in the TDK region.Increases of 4 to 10-fold were found in the media with the mutantGCases, the AKD showed the greatest increases and the AAA the least. Theright panel shows the CRIM Specific Activity, relative intrinsiccatalytic activities of the TKD and its alanine substituted mutantvariants. (B) Left Panel: Activities in the media following transienttransfections of various DSPIIV (SEQ ID NO 15) alanine substitutedGCases and the % of GCase secreted. The latter was calculated as {ngGCase (in media)/[ng GCase (in media)+ng (GCase in lysates)]}×100 (alsosee Table 2). The ESPIIV (SEQ ID NO 20) GCase did not alter secretion,i.e., little enzyme in the media. All of the alanine substitutions inthe DSPIIV (SEQ ID NO 15) sequence resulted in ˜20-25 fold increase inGCase activity in the media (solid bars). The transfections alone(Null+lipo) had no effect. In comparison, the amount of secreted GCaseprotein varied between 22 and 83% (hatched bars) Right Panel: Shows theCRIM specific activity of WT and these same mutant GCases demonstratingthat the mutations decreased this activity relative to WT as indicatedabove the bars. (C) Cellular lysate GCase activities from the variousalanine substituted GCases in the DSPIIV (SEQ ID NO 15) sequence asindicated. The WT and ESPIIV (SEQ ID NO 20) had equal intracellularactivities of GCase, whereas the singly alanine substituted GCases had˜30% of WT levels. The doubly alanine substituted GCase had littleintracellular activity, i.e., nearly all the GCase was secreted into themedia.

FIG. 12: Stoichiometry between ldLIMP2 and DSPIIV (SEQ ID NO 15). Thepeptide concentration giving 50% of maximal binding to ldLIMP2(ordinate) and the concentration of ldLIMP2 (abscissa) have a 1:1 ratio.These results indicate a 1:1 stoichiometry and tight binding propertiesof DSPIIV (SEQ ID NO 15) and ldLIMP2.

FIG. 13: Competition and binding of fluorescent labeled or unlabeledDDQRLLL (SEQ ID NO 14) GCase peptides to ldLIMP2. (A) The δmP of the WT(DDQRLLL (SEQ ID NO 14)) or variously alanine substituted labeledpeptides were plotted against the corresponding unlabeled peptidecompetitors. Note that the fixed ldLIMP2 concentration was 1000 nM or 20times that used in similar studies (FIG. 7). For all peptides the δmPwere near or at background levels. (B) The binding δmP for the variouspeptides as in (A). The signals were near background levels for allpeptides using concentrations 5 to 10 times that for DSPIIV (SEQ ID NO15) and with a 20 times greater LIMP2 fixed concentration.

FIG. 14: Immunoprecipitation of GCase in the presence of ldLIMP2 andincreasing molar ratios of the WT (DDQRLLL (SEQ ID NO 14)) peptide. In(A) or (B) ldLIMP2 and DDQRLLL (SEQ ID NO 14) were incubated before (A)or together with (B) GCase and analyzed as in FIG. 8. Under eithercondition, the peptide did not complete with GCase for ldLIMP2 binding.

DETAILED DESCRIPTION

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes reduction of accumulated materialsinside cells of relevant diseases tissues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that when administered to an organism has a biological effect onthat organism is considered to be biologically active. In particularembodiments, where a protein or peptide is biologically active, aportion of that protein or peptide that shares at least one biologicalactivity of the protein or peptide is typically referred to as a“biologically active” portion. Biological activity can include, forexample, enzyme activity and/or the trafficking activity necessary forbiological function.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form of adisease as the individual being treated, who is about the same age asthe individual being treated (to ensure that the stages of the diseasein the treated individual and the control individual(s) are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from one or more disease as disclosed herein.

Polypeptide, peptide: As used herein, a “polypeptide” or “peptide”,generally speaking, is a string of at least two amino acids attached toone another by a peptide bond. In some embodiments, a peptide mayinclude at least 3-5 amino acids, each of which is attached to others byway of at least one peptide bond. Those of ordinary skill in the artwill appreciate that peptides sometimes include “non-natural” aminoacids or other entities that nonetheless are capable of integrating intoa polypeptide chain, optionally.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains. Substitution of one aminoacid for another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein or peptide that confers a therapeutic effect on the treatedsubject, at a reasonable benefit/risk ratio applicable to any medicaltreatment. The therapeutic effect may be objective (i.e., measurable bysome test or marker) or subjective (i.e., subject gives an indication ofor feels an effect). In particular, the “therapeutically effectiveamount” refers to an amount of a therapeutic protein, peptide, orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset of the disease, and/or also lesseningthe severity or frequency of symptoms of the disease. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular therapeutic protein, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents.Also, the specific therapeutically effective amount (and/or unit dose)for any particular patient may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific pharmaceutical agent employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration,and/or rate of excretion or metabolism of the specific fusion proteinemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic protein (e.g.,GCase or a peptide as described herein) that partially or completelyalleviates, ameliorates, relieves, inhibits, delays onset of, reducesseverity of and/or reduces incidence of one or more symptoms or featuresof a particular disease, disorder, and/or condition (e.g., a lysosomalstorage disease such as Gaucher disease or a neurodegenerative disease).Such treatment may be of a subject who does not exhibit signs of therelevant disease, disorder and/or condition and/or of a subject whoexhibits only early signs of the disease, disorder, and/or condition.Alternatively or additionally, such treatment may be of a subject whoexhibits one or more established signs of the relevant disease, disorderand/or condition.

The peptide sequences on GCase and others like it that target the enzymeto the lysosome have not been elucidated prior to Applicant's disclosureherein. Here, Applicant has identified the peptide sequence on maturehuman GCase that is the ligand for LIMP-2 and has further identifiednovel mutations at specific amino acids that alter the localizationwithin and secretion of GCase from cells.

In particular, disclosed is a new peptide sequence, not previouslyrecognized, on GCase, that is essential for its targeting to thelysosome. Also disclosed are novel and innovative methods for thetreatment of diseases, including lysosomal storage diseases such asGaucher disease and its carrier state, as well as neurodegenerativediseases including Parkinsonism and Alzheimer disease. The connectionsbetween Gaucher disease and these much more common diseases was recentlymade by several groups, including Applicant's (39-42).

Applicant has identified the GCase binding sequence to the luminaldomain of LIMP-2 (“ldLIMP-2”) using heterologous expression of deletionconstructs, the available GCase crystal structures, and by binding andcolocalization of identified peptides or mutant GCases. These studiesshow a complex interaction of ldLIMP-2 with the highly conserved 11amino acid sequence, DSPIIVDITKD (SEQ ID NO: 3), within human GCase. Thebinding is not dependent upon a single amino acid, but its interactionswith LIMP-2 are heavily influenced by D399 (D399E having been identifiedas an equally effective residue, as described below) and thedi-isoleucines, 1402 and 1403. Single alanine substitutions at any ofthese positions decreases binding to ldLIMP-2 and alters their pHdependent ldLIMP-2 binding as well as diminishing GCase's trafficking tothe lysosome while increasing significantly GCase secretion. Thus, bymutation at any or all of these positions, or a combination thereof,decreased GCase binding and increased GCase secretion can be achieved.Replacement of the di-isoleucines and D399 with alanines obliteratedbinding to ldLIMP-2 and lysosomal localization.

However, the retention of the charge at D399, i.e., E399, did not alterthe cellular localization or secretion of GCase. In comparison, alaninereplacements in the TKD sequence (positions 407-409) showed lessprofound effects on ldLIMP-2 binding as single substitutions, i.e., TAD,compared to double, AKA, or triple, AAA, substituted GCases. Finally,the lack of trafficking to the lysosome of the deletion constructGCase-150 that contains the DDQRLLL (SEQ ID NO 14) sequence and the lackof binding of DDQRLLL (SEQ ID NO 14) peptides to ldLIMP-2, eliminatesthis known indirect lysosomal trafficking signal from participation inGCase targeting. This is a surprising result, in that DDQRLLL (SEQ ID NO14) includes a known motif for lysosomal targeting of transmembranelysosomal proteins and is highly conserved throughout phylogeny.

The GCase 11 amino acid ligand for LIMP-2 identified by Applicant ishighly conserved throughout phylogeny with near identity at 10 of theamino acids. T407 of the human GCase has an alanine substitution inseveral mammalian lysosomal GCases so that the consensus sequence across60 species including some insects and worms is DSPIIVDIAKD (SEQ ID NO13). Also, only in mosquito does the E399 equivalent occur, though, asstated above, the D399E mutant has binding properties identical to thatof the human WT sequence. The IIV motif is identical across most of thespecies and in the few with variation, the branch chain amino acid V issubstituted for either of the I, usually the 1402 equivalent.

Enterovirus 71 causes foot and mouth disease by gaining access to cellsafter binding to LIMP-2/SCARB2 (30-32). The binding sequence onEnterovirus 71 for LIMP-2/SCARB2 has been localized to between VP1 aminoacids 152 and 178 (30), but BLAST searches with the entire virusrevealed no significant similarity to GCase and specifically in the 11amino acid region. Several other proteins, e.g., KCNQ1, KCNE2, andMegalin, interact with LIMP-2/SCARB2 at the plasma membrane (33), butthese do not share any homology with GCase. Other intracellular proteinsthat bind to LIMP-2 have not been identified (33). Because theLIMP-2/SCARB2 binding sequences for Enterovirus 71 and GCase are notsimilar, LIMP-2/SCARB2 may have a structure that contains multiplebinding sites with differing specificities. Such structures are presentin other receptors for carbohydrates, i.e., the macrophage mannosereceptor that contains multiple carbohydrate binding domains withdiffering affinities/specificities for mannosyloligosaccharides (43,44). Also, the cationic independent mannose-6-phosphatereceptor/insulin-like growth factor receptor 2 has differingspecificities for mannose 6-phosphate containing glycoproteins andprorenin (8, 45). Indeed, the residues on LIMP-2/SCARB2, which arecritical to GCase binding, are within the same large segment of thereceptor (residues 142-204) that may interact with a “canyon” for thevirus and potentially the sequence of GCase binding. LIMP-2/SCARB2residue H171 in this region appears critical to GCase binding, whereasamino acids 144-151 are essential for Enterovirus 71 binding andinfectivity (30). Consequently, the region of binding for these twodisparate proteins with different ligand sequences occurs in a regionwith overlapping, but apparently distinct, critical residues.

Such specificities are important for understanding the biology of LIMP-2as are the other intracellular proteins that may bind to LIMP-2/SCARB2.For example, mutations in LIMP-2 are causal to a human and mousedisease, termed action myoclonus-renal failure syndrome (AMRF) (34). Tounderstand the biochemical bases of this disease, it will be critical tounderstand which proteins interact with LIMP-2/SCARB2 and cause AMRFrather than Gaucher disease-like manifestations. However, being aheterozygote for a pathogenic LIMP-2/SCARB2 mutation potentiates thephenotype in Gaucher disease homozygotes (46) Additionally, disruptivemutations of GBA1 in the sequence critical to LIMP-2/SCARB2 may havemore profound effects on the Gaucher disease phenotype then might beanticipated by the in vitro levels of residual activity, as the mutantprotein may never reach the lysosome. The biology of LIMP-2/SCARB2 alsomay facilitate the understanding of the clear relationships betweenParkinson disease and Lewy Body dementia. Indeed, the role of GCasebinding to LIMP-2/SCARB2 in the ER may be important to the proposed feedforward mechanism of GCase/α-synuclein interaction (40) in thedevelopment and treatment of Parkinson disease and otherα-synucleinopathies, such that interference of this binding may reverseor otherwise attenuate development of α-synucleinopathy disease states.

The precise role of LIMP-2 in trafficking GCase remains unexplored, butthe studies here and in LIMP-2 deficient patients and mice offer someinsights. GCase activity does not depend on whether it binds to LIMP-2.The poor or deficient binding of GCase variants to LIMP-2 in thesestudies does not appear to alter the enzyme activity or stability ofGCase. Also, the LIMP-2 knock-out mouse cells produce active GCase,albeit at lower intracellular levels that WT (23), because of secretionfrom the cells. These findings suggest that, like the M6PR, LIMP-2 doesnot have a chaperone/protective function. LIMP-2/GCase binding also isnot needed for GCase stability vis-à-vis saposin C's stabilization ofGCase against proteolysis (47). Thus, LIMP-2/SCARB2's function is todeliver intracellularly synthesized GCases to the lysosomes.

The identification of a critical sequence for GCase binding to itsreceptor, LIMP-2/SCARB2 may have significant therapeutic implications.These studies indicate that GCases with specific mutations that disruptbinding to its receptor, but not its activity, could provide forenhanced secretion of GCase from cells for bulk production in selectedcells. Since such mutations could greatly enhance the secretion ofactive enzyme suggesting the potential for enhanced production frommammalian or other systems that contain LIMP-2/SCARB2analogues/homologues. Similarly, transplantation of specific cell typescontaining specifically expressing mutated GCases that interact poorlywith LIMP-2, could supply large amount of secreted enzyme fortherapeutic effects both in local and distant parts of the body. Forexample, for an enzyme that is normally not secreted, such secretion,attained by cellular replacement and/or gene therapy approaches withcontrollable expression elements, could provide a reservoir for thesupply of secreted, active enzyme for cross correction in other cells.Such an approach would be particularly helpful in the CNS variants ofGaucher or Parkinson diseases, which are caused by or potentiated byGCase defects (48, 39) or the consequences of α-synucleinopathies (38,49-51), and generalized distribution of the enzyme throughout the brainis currently not possible.

Modified GCase Protein (Recombinant Protein)

In one aspect, GCase proteins comprising one or more mutations aredisclosed. By mutating the amino acid sequence in the wild-type GCaseprotein (SEQ ID NO:2), production of the recombinant protein may besignificantly increased. This enzyme is not normally secreted from cellsand, therefore, wild type enzyme produced is retained and is notavailable in the surrounding media for purification. Mutation of theGCase with retention of full activity can be accomplished and many foldgreater levels of the enzyme can be recovered from the media as apotential therapeutic by site-specific mutation of the WT GCase proteinas described herein. This has major advantages over existing approachesfor the production of such proteins, allowing for improved methods ofproducing GCase which can subsequently be used to treat or reduce theeffects of a variety of disease states as described herein.

In one aspect, disclosed herein are GCase proteins comprising one ormore mutations that may comprise from about 85%, or from about 90%, orfrom about 95%, or from about 96%, or from about 97%, or from about 98%,or from about 99% sequence homology to human GCase sequence (SEQ ID NO2), wherein the recombinant protein may comprise a mutation at one ormore positions as described herein. In one aspect, the position whereina mutation may occur is selected from position 397, 399, 400, 401, 402,403, 408, 409, 410, and a combination thereof, with reference to SEQ IDNO 2. The one or more mutations decrease LIMP-2 binding, or in otheraspects, substantially ablates LIMP-2 binding. In some aspects, themutation may decrease binding by about 10%, or about 20%, or about 30%,or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, orabout 90% or about 100% as compared to WT binding. In one aspect, themutation is such that the change in the amino acid changes the charge atthe one or more mutated positions. In one aspect, the change is to apositively charged amino acid. In one aspect, the mutation may changethe amino acid at the mutated position to an amino acid selected fromalanine and glycine. In one aspect, the mutation may change the aminoacid at the mutated position to alanine. It should be noted that, withrespect to position 399, aspartate (D) may be mutated to glutamate (E)without effect to the binding. One of ordinary skill in the art willreadily appreciate that certain amino acid substitutions are likely tobe conservative in nature, such that a mutation at any one particularresidue that is anticipated to be a conservative mutation is likely tohave minimal effect on structure or function. As such, apart from and inaddition to the described mutations above, Applicant notes thatsubstantially homologous GCase proteins as defined above are within thescope of the disclosure.

In one aspect, the GCase protein may comprise a mutation at a positionselected from 399, 402, 403, 407, 408, 409, and a combination thereofwith reference to SEQ ID NO 2. In another aspect, the mutation may occurat a position selected from 399, 402, 403, and a combination thereofwith reference to SEQ ID NO 2. In one aspect, positions 399-403 may bemutated such that this region of the sequence comprises a sequenceselected from ASPII (SEQ ID NO 7), DSAII (SEQ ID NO 8), DSPAI (SEQ ID NO9), DSPIA (SEQ ID NO 10), ASPIA (SEQ ID NO 11), ASPAA (SEQ ID NO 12).Similarly, the mutation may include any of the above sequences andfurther include the mutation D399E.

In one aspect, the mutation in the GCase protein may occur at a positionselected from 407, 408, 409, and a combination thereof, with referenceto SEQ ID NO 2. In one aspect, the amino acid sequence at positions407-409 of SEQ ID NO 2 may comprise a sequence selected from AAA, AKA,and TAD. Again, it is to be recognized that D at position 409 may bemutated to E (glutamate) at position 409.

In one aspect, the GCase protein may comprise a mutation that decreaseslocalization of the recombinant protein to the lysosomes of a cell whileit is synthesized. In one aspect, the GCase protein comprising one ormore mutations as described above may have biological activity and/orenzymatic activity that is substantially equivalent to that of wild typeGCase. In one aspect, the GCase protein comprising one or more mutationsmay be capable of being taken up into cells via non-LIMP-2 mechanisms insubstantially the same manner as wild type GCase. The GCase proteincomprising one or more mutations may further comprise additionalN-terminal and/or C-terminal amino acids. See, for example, variantsdisclosed in Brumshtein, B., et al. (2006). “Structural comparison ofdifferently glycosylated forms of acid-beta-glucosidase, the defectiveenzyme in Gaucher disease.” Acta Crystallogr D Biol Crystallogr 62 (Pt12): 1458-1465, including a carrot cell variant, and a CHO cellexpressed GCase comprising an amino acid arginine to histidine mutationat residue 495 (R495H). In one aspect, the GCase protein comprising oneor more mutations as described herein may increase secretion of therecombinant protein or peptide from cells in which it is synthesized. Itshould be noted that the R495H mutant noted above is not believed toincrease secretion as a result of the R to H mutation, but rather,secretion is likely due to heavy overexpression of the protein andsaturation of LIMP-2.

In one aspect, the GCase protein comprising one or more mutations mayprovide significant advances for treatment. For example, the transformedand transplanted hematopoietic stem cells or transduced hepatic cellsmay be enabled to secrete large amounts of GCase that would function asan enzyme therapy in addition to the RES replacement via thehematopoietic system. These cells could be altered by, for example, insitu “gene therapy” by delivery of the gene therapeutic to the maturecells from the recipient or by use of induced pluripotent stem cell(iPSC)-derived progenitors that are genetically altered and returned tothe same person from which the cells were derived, i.e., the donor andrecipient are the same person.

In one aspect, the GCase proteins comprising one or more mutationsdescribed herein may be modified for use in neurological diseases inwhich gene therapy with GCase would be beneficial, i.e., Parkinson orAlzheimer diseases. For example, a gene therapy vector, e.g., AAV9/10serotypes, for delivery of the cDNA for GCase to the striatal and/orhippocampal areas and the altered enzyme would be secreted in largeamounts and result in metabolic correction of neighboring cells. In thiscase and in the case of using mutated GCase in hematopoietic stem cellsor hepatic cells as described above, the enzyme may be taken up by otherreceptors because the peptide sequence does not participate inreceptor-mediated endocytosis. Similarly, iPSC-derived neuronalprogenitor cells (NPCs) could be genetically altered with thetherapeutic hepatic cells and/or hematopoietic stem cells as describedabove, and then be used for intraventricular and/or stereotacticintracerebral injections. The former would require migration of the NPCsto areas of damage and the latter would require direct injection of theNPCs into areas of involvement. These approaches would lead to bothcellular and enzyme reconstitution in the damaged brain.

Peptides

In one aspect, peptides are disclosed, wherein the peptide can be usedas a therapeutic independently, or to facilitate any of theabove-described methods without having to mutate the wild-type GCase.For example, the disclosed peptide would compete for the known receptorfor this enzyme, LIMP2, and not allow the enzyme to be targeted to thelysosome, but rather to be shunted to the secretory pathway in largeamounts.

In one aspect, peptides comprising residues 347 through 422 of SEQ ID NO2 are disclosed. The peptides may be further combined with apharmaceutically acceptable excipient for administration to a subject.In one aspect, the peptide binds to a LIMP-2 receptor. It should benoted that this is different than the enterovirus sequence that binds toLIMP-2. In one aspect, the peptide may comprise amino acids 397 through409 of SEQ ID NO 2, or amino acids 399 through 403 of SEQ ID NO 2, oramino acids 407 through 409 of SEQ ID NO 2. Again, it is to berecognized that position 399 may be mutated to D399E. Likewise,aspartate (D) residues in the peptide sequences may generally be mutatedto glutamate (E) residues while still preserving the binding of thepeptide to a LIMP-2 receptor. The peptides described herein may preventlocalization of GCase to a lysosome of a cell in which GCase is beingsynthesized and/or may increase secretion of endogenous GCase.

In some aspects, the peptides may comprise from about 3 to about 400, orabout 3 to about 300, or about 3 to about 200, or about 3 to about 100,or about 3 to about 50, or about 3 to about 25, or about 3 to about 10amino acid residues. In one aspect, the peptide may be a syntheticpeptide of any length capable of being produced using methods known inthe art. It will be appreciated that varying lengths of peptidesequences will be suited for administration as described herein, andthat modification and manufacture of such peptides is well within theordinary skill in the art.

DNA Vectors

In one aspect, DNA vectors capable of expressing a recombinant proteinand/or peptide as described above are disclosed. Suitable vectors mayinclude viral vectors, (including lenti, retro, AAV, Adeno, Herpes,rabies, any viral vector), plasmid vectors, cosmid vectors, or any otherconstruct capable of expressing GCase proteins in cells containingLIMP-2 as will be readily understood by one of skill in the art.

Cell Lines

In one aspect, a cell or cell line that may comprise a DNA sequencecapable of expressing a GCase protein comprising one or more mutationsand/or a peptide as described above is disclosed. Cells suitable for usewith the present disclosure will be readily apparent to one of ordinaryskill in the art. For example, the cell may be selected fromfibroblasts, or any other cell capable of producing the above-describedproteins or peptides, including, for example, any nucleated cell, moreparticularly, CHO cells, human fibrosarcoma cells, carrot cells, avertebrate cell that expressed LIMP-2, a hematopoietic stem cell, atransduced hepatic cell, a neuron, a microglial cell, or an inducedpluripotent stem cell (iPSC)-derived progenitor. The cells may betransiently or stably transfected/transformed.

Methods of Treating Disease

In one aspect, one or more of the above-described GCase proteins orpeptides may be used to treat one or more disorders or disease states ina subject in need thereof.

In one aspect, a method of inhibiting lysosomal LIMP2 is disclosed,wherein the method may comprise the step of administering one or moretherapeutic agents selected from a GCase protein comprising one or moremutations as disclosed herein; a peptide as disclosed herein; a DNAvector as disclosed herein; a cell that produces a GCase proteincomprising one or more mutations and/or a peptide as disclosed herein;or a combination thereof, to a patient in need thereof.

In one aspect, a method of treating a disorder related to a dysfunctionin the GCase pathway is disclosed, the method comprising the step ofadministering one or more therapeutic agents selected from a GCaseprotein comprising one or more mutations as disclosed herein; a peptideas disclosed herein; a DNA vector as disclosed herein; a cell thatproduces a GCase protein comprising one or more mutations and/or apeptide as disclosed herein; or a combination thereof, to a patient inneed thereof.

The disorders contemplated in the methods disclosed may comprise adisorder related to defective GCase activity and/or decreased enzymaticactivity. In other aspects, the disorder may comprise a disorderassociated with alpha-synuclein dysregulation. The disorder may comprisea lysosomal storage disease, for example, Gaucher disease, Fabrydisease, Pompe disease, mucopolysaccharidoses, and multiple systematrophy. In other aspects, the disorder may comprise a neurodegenerativedisorder, for example, Parkinson disease, Alzheimer disease, or Lewybody dementia. In one aspect, the disorder may comprise a dysfunction inthe GCase pathway that results in a central nervous system diseasewherein the pathogenesis of said disease results in plaques and/ordisease states associated with plaques.

The methods may employ a variety of different routes of administration.In one aspect, the administration step may be selected fromadministration via intraventricular intracerebral injection,stereotactic intracerebral injection, intravenously, orally,subcutaneously, rectally, intranasally, via inhalation, and combinationsthereof. In one aspect, for example, the method may comprise the step ofadministering one or more therapeutic agents as described herein viaintraventricular and/or stereotactic intracerebral injection.

In a further aspect, the administration step may comprise isolation of acell from a patient in need of treatment; introducing into the cell ofthe patient a sequence selected from a sequence capable of expressing aGCase protein comprising one or more mutations as disclosed herein,and/or a sequence capable of expressing a peptide as disclosed herein;and reintroducing the cell into the patient. In this aspect, the cellmay then produce the GCase protein comprising one or more mutationsand/or a peptide as disclosed herein.

Examples

Materials

The following were from commercial sources:4-methylumbelliferyl-β-D-glucopyranoside (4MU-Glc; Biosynth AG,Switzerland); Sodium taurocholate (Calbiochem, La Jolla, Calif.); Rabbitanti-LIMP2 polyclonal antibody, rabbit anti-LAMP1 antibody, Goatanti-actin antibody (Santa Cruz BioT., Dallas, Tx); goat or rabbitanti-Calreticulin, -calnexin antibodies (Abcam, Cambridge, UK); NuPAGE4-12% Bis-Tris gel, NuPAGE MES SDS running buffer, DMEM, pBluescriptvector, Dynabeads protein G Immunoprecipitaion Kits, BS3 chemicalcrosslinker (Invitrogen, Carlsbad, Calif.); BCA Protein Assay Reagent(Pierce, Rockford, Ill.); pCMV-AC-GFP/YFP/cMyc expression vectors(Origene, Rockville, Md.). PVDF membranes and ECL detection reagent(Amersham Biosciences, Piscataway, N.J.); ABC Vectastain and AlkalinePhosphatase Kit II (Black) (Vector Laboratory, Burlingame, Calif.).Restriction enzymes (New England Biolabs Inc., MD); site-directedmutagenesis kits (Clontech Lab. Inc., Mountain View, Calif. orQuikChange, Stratagene, Tex.). Purified luminal domain LIMP-2 (1d-LIMP2)was custom made (Sino Biological Inc., PRC.) Imiglucerase™ was a giftfrom Genzyme Corp. a Sanofi Company, Cambridge, Mass. Rabbit anti-GCasepolyclonal antibody was produced in this laboratory (Fabbro D, andGrabowski G A. Human acid beta-glucosidase. Use of inhibitory andactivating monoclonal antibodies to investigate the enzyme's catalyticmechanism and saposin A and C binding sites. J Biol Chem. 1991;266(23):15021-7).

Methods:

Deletion constructs of GCases: The full-length human GCase cDNA inpBluescript was used as backbone for deletion constructs. Four singlecut Restriction enzymes (ScaI, BstAPI, BalI and BamHI) were used todigest the full-length (FL) cDNA to created these deletion constructs(GCase-225, GCase-150, GCase-75 and GCase-23). These were subsequentlyindividual clones into pCMV6-AC-GFP vector for mammalian cell expressionto provide GCase-XX in frame with GFP. All constructs were resequencedfor verification.

GCase expression constructs for point mutations: Using site-directedmutagenesis kits and designed primers, all single amino acid mutationconstructs were created using the full Length pCMV-GCase-GFP or YFPvectors. All constructs were sequenced for verification and no oradditional mutations were found.

Immunoprecipitation of GCases with mutations in the TKD sequence:

For immunoblots, Gba1 knockout mouse fibroblasts (null/null) weretransfected with the GBA1 constructs (WT or mutants) and expressed for 5days. The GCases from harvested cell lysates were purified using a GCaseantibody affinity column (Dynabeads protein G crosslinked (BS3) withrabbit anti-human GCase antibody). The purified GCases were then mixedwith LIMP2 (1:1), incubated (30 min.), and applied to another affinitycolumn (Dynabeads protein G crosslinked (BS3) with rabbit anti-LIMP2antibody). Aliquots of each step (loading, wash and eluent) werecollected and analyzed on 12.5% SDS gels. Immunoblots were developedusing anti-human GCase antibody and AP conjugated color developmentkits. Mock experiments with either non-LIMP2 proteins added or no GCasesadded, were used as control. The quantitations of theseimmunoprecipitation data were obtained from densitometry measurements ofthe gels. The no-LIMP-2 bars are the controls for comparison, sinceGCase was processed through all steps without LIMP-2 to account for anylosses of GCase; About 50% of the GCase was lost during processing.

Fluorescence Polarization: The DSPIIV (SEQ ID NO 15) and its mutants,and DDQRLLL (SEQ ID NO 14) peptides (FITC labeled or non-labeled) weresynthesized (American Peptide Co., Vista, Calif.). Lyophilized peptideswere solubilized in acetonitrile and then were diluted at least10,000-fold into the reactions for fluorescence polarizationassessments. The reactions were done at least in duplicate at variouspeptide and LIMP-2 concentrations (SpectraMax M5, Molecular Device).Fluorescence polarization data were collected and analyzed with SoftMaxPro 5.0 software. Reaction mixtures contained 100 mM phosphate, pH 6.8and 1 mM DTT.

Immunofluorescence studies (IF): Mouse Gba1 null/null were used for thehost cells for all transfection experiments. pCMV-AC-GFP Mammalian cellexpression vector was used for expression of various GCases thatincluded the TKD region (amino acids 407, 408 and 409) or the DSPIIV(SEQ ID NO 15) region (amino acids 399-404). Direct-labeled firstantibodies were applied for IF detection; including anti-human GCase,-Calreticulin, -calnexin, -LIMP2, and -Lamp1. Lysotracker and ER tracker(Invitrogen) were also used as indicated. Analyses were done with aZeiss AxioVert 200M with ApoTome. Co-localization analyses to obtainPearson Indices were conducted using a module in the Axiovision 4.8software.

Protein sequence homologies were performed using MUSCLE and BLAST (52,53)

Enzyme Assays: Cell pellets were homogenized in 0.25% Na-taurocholateand 0.25% Triton X-100. GCase activities were determinedfluorometrically with 4 mM 4MU-Glc in 0.2M/0.1M citric-phosphate, pH 5.6(18, 54)

Results

Initial studies to localize the targeting peptide were conducted withGCase-GFP fusion constructs that had COOH-terminal deletions of GCase(FIG. 1). The constructs are designated as GCase-XX or XXX where the Xsrepresent the number of GCase amino acids deleted from theCOOH-terminus. These constructs were transfected into and expressed inGba1 null/null mouse fibroblasts using the CMV promoter (pCMV-AC-GFP orYFP) Immunofluorescence analyses using anti-human GCase IgG (green) andLysotracker (Red) were used for colocalization of GCase to the lysosome(Red) or GCase (green) and Calreticulin or Calnexin (Red) forcolocalization to the ER or Golgi (FIG. 2). The Pearson Indices forthese immunofluorescence studies indicated that GCase-23 and GCase-75had similar colocalization to the lysosome as full length WT GCase. Incomparison, GCase-150 and GCase-225 were more localized to the ER andGolgi, and much less to the lysosome with Pearson Indices similar to theGFP control without GCase sequences (N-GFP). These studies showed thatthe critical GCase amino acids for targeting to the lysosome werebetween amino acids 347-422, i.e., in the fragment between GCase-75 andGCase-150. Since LIMP-2 is the receptor for targeting of GCase to thelysosome, our working hypothesis was that the peptide sequence forbinding of GCase to LIMP-2 was located within amino acids 347-422.

The location of this GCase amino acid sequence is shown in a linear formin FIG. 3 (bracketed and in Orange highlight). This amino acid sequenceis located in Domain I of the GCase crystal structure. Analysis of thisregion shows that the amino acids between N399 and D409 are surfaceexposed and form a structure that would be accessible for ligand bindingto LIMP-2 (FIG. 3). However, amino acids F347-D398 and F411-H422 aremore internal to the structure and less likely to be able to interactwith LIMP-2. The likely exposed residues include the sequenceDSPIIVDITKD (SEQ ID NO 3) (FIG. 4, in yellow). Searches of the databasesfor lysosomal glucocerebrosidases from multiple species show that thissequence is highly conserved and nearly invariant in mammals; theconsensus sequence is DSPIIVDIAKD (SEQ ID NO 13). Based on theseanalyses, efforts were targeted to the amino acid sequence between D399and D409 (FIG. 4). Although GCase is a membrane associated and not atransmembrane lysosomal protein, the mature GCase sequence does containa di-leucine structure that has been previously identified as importantin the indirect targeting of transmembrane proteins to the lysosome(55-57). This sequence spans amino acids D282-L288 (FIG. 3), DDQRLLL(SEQ ID NO 14) (FIG. 4) is also located in Domain III and was alsoanalyzed for interactions/binding to LIMP-2 as a potential ligandsequence.

LIMP-2 is a transmembrane protein whose cytoplasmic domain (ldLIMP-2)binds to and trafficks GCase to the lysosome (23). A series ofalanine-swapped mutant proteins and peptides were made for cellularlocalization, immunoprecipitation, and secretion analyses. For theexpression studies, Gba1 null/null fibroblasts were used and the GCasevariant localized to cellular compartments. A 3 amino acid (TKD) and a 5amino acid (DSPIIV (SEQ ID NO 15)) sequence of the complete 11 aminoacid sequence of interest (FIG. 3) were targeted for alaninemutagenesis. The various mutant GCases for the TKD sequence showedimmunofluorescence lysosomal localization (WT, AKA, TAD) to varyingdegrees or lack of this property (AAA), i.e., the AAA mutant showed nolysosomal localization, but abundant colocalization to the ER by usinganti-calnexin/calreticulin. Thus, AAA was expressed in the cell, but wasnot trafficked to the lysosome. However, the WT and TAD mutant hadsimilar ER and lysosomal localizations (FIG. 5). The AKA mutant showedless lysosomal localization compared to WT or the TAD mutant (FIG. 5).

To demonstrate a correlation between the colocalization results andLIMP-2 binding, the WT and variant GCases were expressed and purified byanti-GCase affinity chromatography, and used to conduct in vitroimmunoprecipitation studies with ldLIMP-2 (FIG. 6). These results showthat WT and the TAD GCases (FIGS. 6 A and D) were bound similarly byldLIMP-2, whereas the AKA mutant (FIG. 6C) was much less bound and theAAA GCase did not bind to ldLIMP-2 (FIG. 6B). Quantitative densitometryresults are shown in FIG. 6E. These results for ldLIMP-2 bindingcorrelated well with the colocalization analyses, which followed asimilar pattern for the WT, intermediate, or absent lysosomallocalization.

Several additional peptides were used to explore the DSPIIVDITKD (SEQ IDNO 3) region by alanine scanning mutagenesis (Table 1) and a peptidefrom another region of GCase was used as a control. These peptides weresynthesized with and without a fluorescent probe (FITC) covalentlylinked to the C terminal end to assess direct binding to ldLIMP-2 bychanges in fluorescence polarization.

TABLE 1 Synthesized Peptides Sequence location M.W. (*FITC) M.W.(nonlabeled) Purity (%) DSPIIV 399-404 1032.2 642.8 95.3/98.0 ASPIIV 399-404998.1 578.8 95.0/98.1 DSPAIV 399-404 990.1 600.7 96.5/98.2 DSPIAV399-404 990.1 600.7 95.3/97.7 DSPAAV 399-404 948.1 514.6 95.4/95.1SKDVPL 465-470 1047.2 657.8 96.6/98.0 DDQRLLL 282-288 1261.4 87295.3/97.1 ADQRLLL 282-288 1226.4 828 96.1/97.6 DAQRLLL 282-288 1226.4828 95.7/97.1 DDQRAAA 282-288 1144.2 745.8 95.9/98.0 DFIARDL 258-2641247.4 849 95.1/97.5 Peptides were custom synthesized by AmericanPeptide Company QC

 HPLC grade, purity: 95.1-98.8%. MS analysis/desalted/lyophilized intopowder. Peptides were solubilized in Acetonitrile and further diluted inexcessive water (>10000-fold) with sonication. Product manufac. Numberstarted from 35310 to 35316 and 35340 to 35346.

indicates data missing or illegible when filed

The interactions/binding of the GCase peptides, DSPIIV (SEQ ID NO 15)variants, to ldLIMP-2 are shown by the change in fluorescencepolarization with increasing WT or mutant peptide concentration in thepresence of a fixed amount of ldLIMP-2 (FIG. 7A). The substitution of analanine for either of the isoleucines decreased binding to ldLIMP-2 byabout 50%, whereas alanine substitution for the N-terminal aspartatenearly eliminated the binding to ldLIMP-2. Substitution of alanines forD399, 1402 and 1403 obliterated the binding.

To ensure that the fluorescent label was not interfering or promotingbinding to ldLIMP-2, a similar experiment was conducted by addition ofan unlabeled peptide to compete with the corresponding labeled peptide.The data show direct competition of the corresponding labeled andunlabeled peptides (FIG. 7B). Similarly, a direct comparison wasconducted to assess the competition of either the labeled or unlabeledWT peptide for binding to ldLIMP-2 (FIG. 7C). The data show a symmetryof the change in fluorescence polarization that are nearly mirror imageswith either the labeled or unlabeled WT peptide in competition withldLIMP-2. These data show that the fluorescent label did not alter thebinding properties to ldLIMP-2. Using the WT labeled peptide the peptidebinding to ldLIMP-2 was shown to have 1:1 stoichiometry and tight, i.e.,the concentration of peptide needed for 50% binding to ldLIMP-2 was in a1:1 molar correspondence (FIG. 12).

In other binding experiments, the DDQRLLL (SEQ ID NO 14) labeled andunlabeled peptides were synthesized and used. This peptide had little orno binding to LIMP-2 above background. Thus, the DDQRLLL (SEQ ID NO 14)had essentially no interaction with LIMP-2 (FIG. 13).

Immunoprecipitation and Competition Studies:

To further evaluate the interaction of ldLIMP-2, WT GCase, and theDSPIIV (SEQ ID NO 15) peptide, competitive immunoprecipitationexperiments were conducted using increasing concentrations of the WTpeptide in competition with GCase for binding (FIG. 8A). Increasing WTpeptide, DSPIIV (SEQ ID NO 15), concentrations from 0 to 5× molar excessover GCase showed that the this peptide competes off binding toldLIMP-2, when GCase was first incubated with LIMP-2 and then the WTpeptide was added. A similar experiment was conducted in which the WTpeptide was preincubated with ldLIMP-2 prior to adding purified GCase(FIG. 8B). Almost all GCase binding to LIMP 2 was prevented at a 1 to 5×molar peptide ratios. The combined results (FIGS. 8A and B) show thatthe WT peptide can compete or prevent GCase binding to ldLIMP-2.

The DDQRLLL (SEQ ID NO 14) peptide from GCase was tested as a control.Although this sequence was contained in the GCase fragment (GCase-225)that did not localize to the lysosome, it does have a known motif forlysosomal targeting of transmembrane lysosomal proteins, which GCase isnot. In addition, this sequence is highly conserved throughoutphylogeny. Up to a 5× Molar excess this peptide does not compete withGCase for ldLIMP2 binding when the peptide was preincubated with LIMP-2before GCase was added or when LIMP-2, the peptide and GCase wereincubated together. FIGS. 14A and 14B.

pH-Dependency of ldLIMP-2/GCase binding: LIMP-2 binding to GCase isdependent upon pH in that greater binding is obtained at more neutral pHand dissociation obtains at pH 5.6 (23). The WT and various mutants ofthe DSPIIV (SEQ ID NO 15) peptide have differential pH dependency.Substitution of either isoleucine with alanine decreases the binding atpH 6.8 by 40-50%. The ASPIIV peptide (SEQ ID NO 16) binding wasdecreased by about 30-40%, whereas the ASPAAV peptide was decreased by˜90%. There is little effect on the binding at pH 5.8 of any of thepeptides compared to WT (FIG. 9).

Effects of Mutations of DSPIIVDITKD (SEQ ID NO 3) on CellularColocalization and/or Secretion of GCase:

The effects on cellular localization of GCase from cells transfectedwith WT or various GCases with mutations in the target region are inFIG. 10A. Typical examples show that the WT or D399E GCases are targetedto the lysosomes indicating that the charge on amino acid 399 isimportant for this targeting. In comparison, the DSPAIV (SEQ ID NO 17)(1402A) or DSPIAV (SEQ ID NO 18) (I403A) do not localize to thelysosome. Essentially identical results were obtained with the ASPIIV(SEQ ID NO 16) (D399A) or 402A/I403A (double mutant) GCases.Confirmatory quantification by Pearson Indices of colocalization to theER/Golgi or lysosome of all these expressed WT and mutant GCases are inFIG. 10B.

Corresponding effects on the secretion of GCases from the cellscontaining various mutations in DSPIIVDITDK were found by assessing theactivity of GCases in the media and cell lysate (FIG. 11 A-C). Comparedto the WT sequence (TDK), the amount of GCase activity in mediaprogressively increased from the AAA to AKA to TAD mutants to about an8-fold increase (FIG. 11A, left panel). The lower GCase activity inmedia of the AAA mutant seemed at odds with the lack of binding of thismutant to LIMP2 Immunoblots of the various mutations in the TKD sequenceprovided assessments of the GCase activity/amount of CRIM or CRIMspecific activity, an estimate of the catalytic rate constant relativeto WT enzyme. Using this assessment, the ratio of CRIM specificactivities relative to WT (assigned 100%) for AAA, TAD, and AKA were20%, 32%, and 49%, respectively (FIG. 11A, right panel), or that moreGCase protein was required from the respective mutants to achieve thesame enzyme activity as the WT. These results indicate that the levelsof GCase protein secreted from the Gba1−/− fibroblasts were 2.5 to 5fold greater than suggested by the enzyme activity measures andcorrespond well with the binding data (FIG. 6). Similarly for the DSPIIVsequence (SEQ ID NO 15), all of the alanine mutant GCases showed largeincreases (˜25-fold) in media activity compared to WT, i.e., increasedsecretion of active GCase (FIG. 11B, left panel). When assessed for CRIMspecific activity (CRIM SA) the alanine mutants had 14% to 29% of WTactivity, implying that 7.1 (for the double mutant) to 3.4 (I403A) moreGCase enzyme protein was secreted than suggested by the raw activitymeasures. The Gba1−/− cells transfected with the charge conservedsubstituted GCase, D399E, showed no media activity above background(i.e., zero) and a minor change in CRIM specific activity. Using theCRIM SA assessments and the total activities in lysates and media, thepercent secreted GCase (ng) showed that ˜83% of the 1402A/I403A mutantwas secreted from transfected cells. For the other mutants this rangedfrom 22-36%. This compares to ˜0% for the WT or D399E GCasesImmunoaffinity anti-GCase column purification of the various GCases frommedia confirmed the excesses of these mutant GCases and the inability todetect WT or D399E GCases in media (data not shown). Complementary datafor the retention of GCases in cell lysates showed ˜15-20% of WT orESPIIV (D399E) activities with the single alanine mutants [ASPIIV (SEQID NO 16) (D399A), DSPAIV (SEQ ID NO 17) (1402A), and DSPIAV (SEQ ID NO18) (I403A)], and <5% with the double DSPAAV (SEQ ID NO 19)(1402A/I403A) (FIG. 11C). Thus, all of the alanine mutated GCases thatwere shown to diminish ldLIMP-2 binding and poor lysosomal localizationshowed large increases in activity of GCase in the media, i.e., excesssecretion.

TABLE 2 Proportion of GCase protein in cells and media GCase CellularMedia GCase Total GCase Variant GCase (ng) (ng) (ng) % Secreted Human WT23 ± 4 0 23 ± 4 0 D399A 32 ± 4 18 ± 2 50 ± 4 36 I402A 38 ± 6 11 ± 4 49 ±5 22 I403A 34 ± 2 10 ± 2 44 ± 3 23 I402A/I403A  4 ± 1 20 ± 3 24 ± 4 83Mouse WT 64 ± 7 0 64 ± 7 0 Human WT and variant GCases were transfectedinto Gba1−/− cells with resultant transfection efficiencies of 20.5 ±3%. The calculations were based on the specific activities of GCaserelative to a standard curve of cross-reacting immunological material(CRIM) densities on immunoblots of purified human GCase, which has aspecific activity of 1.1 nmole/hr/ng GCase. Cellular and Media GCaserepresent total ng from each source. Results are expressed as the mean ±SD, n = 3.

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Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. Claims or descriptionsthat include “or” between one or more members of a group are consideredsatisfied if one, more than one, or all of the group members are presentin, employed in, or otherwise relevant to a given product or processunless indicated to the contrary or otherwise evident from the context.The invention includes embodiments in which exactly one member of thegroup is present in, employed in, or otherwise relevant to a givenproduct or process. The invention also includes embodiments in whichmore than one, or all of the group members are present in, employed in,or otherwise relevant to a given product or process. Furthermore, it isto be understood that the invention encompasses variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of theclaims is introduced into another claim dependent on the same base claim(or, as relevant, any other claim) unless otherwise indicated or unlessit would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise. Where elements are presentedas lists, e.g., in Markush group or similar format, it is to beunderstood that each subgroup of the elements is also disclosed, and anyelement(s) can be removed from the group. It should it be understoodthat, in general, where the invention, or aspects of the invention,is/are referred to as comprising particular elements, features, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, etc. Forpurposes of simplicity those embodiments have not in every case beenspecifically set forth herein. It should also be understood that anyembodiment of the invention, e.g., any embodiment found within the priorart, can be explicitly excluded from the claims, regardless of whetherthe specific exclusion is recited in the specification.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. Furthermore,where the claims recite a composition, the invention encompasses methodsof using the composition and methods of making the composition. Wherethe claims recite a composition, it should be understood that theinvention encompasses methods of using the composition and methods ofmaking the composition.

All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

To the extent dimensions and values are disclosed herein, such are notto be understood as being strictly limited to the exact numerical valuesrecited. Instead, unless otherwise specified, each such dimension isintended to mean both the recited value and a functionally equivalentrange surrounding that value. For example, a dimension disclosed as “20mm” is intended to mean “about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1.-18. (canceled)
 19. A composition comprising a peptide, wherein saidpeptide comprises an amino acid sequence comprising from about 90% toabout 100% homology to residues 347 through 422 of SEQ ID NO 2, whereinsaid amino acid sequence binds to a LIMP-2 receptor, and wherein saidamino acid sequence is isolated or synthesized.
 20. The peptide of claim19, wherein said amino acid sequence comprises amino acids 397 through409 of SEQ ID NO
 2. 21. The peptide of claim 19, wherein said amino acidsequence comprises amino acids 399 through 403 of SEQ ID NO
 2. 22. Thepeptide of claim 19, wherein said amino acid sequence comprises aminoacids 407 through 409 of SEQ ID NO
 2. 23. The peptide of claim 19,wherein said amino acid sequence inhibits localization of GCase to alysosome of a cell in which GCase is being synthesized.
 24. The peptideof any of claim 19, wherein said isolated amino acid sequence increasessecretion of endogenous GCase.
 25. The peptide of claim 19, wherein saidamino acid sequence comprises from about 3 to about 400 amino acidresidues in length.
 26. The peptide of claim 19, wherein any aspartateresidue is mutated to a glutamate residue, preferably comprising D399E,D405E, D409E, or combinations thereof, more preferably D399E.
 27. Thepeptide of claim 19, comprising the mutation D399E.
 28. The compositionof claim 1 comprising a pharmaceutically acceptable excipient. 29.-35.(canceled)
 36. A method of treating a disorder related to a dysfunctionin the GCase pathway comprising the step of administering one or moretherapeutic agents selected from a. a GCase protein; b. a compositioncomprising a peptide, wherein said peptide comprises an amino acidsequence comprising from about 90% to about 100% homology to residues347 through 422 of SEQ ID NO 2, wherein said amino acid sequence bindsto a LIMP-2 receptor, and wherein said amino acid sequence is isolatedor synthesized; c. a DNA vector capable of expressing a GCase proteincomprising at least about 85% sequence homology to human GCase aminoacid sequence (SEQ ID NO 2) comprising one or more mutations at aposition selected from 397, 399, 400, 401, 402, 403, 408, 409, 410, andcombinations thereof, wherein said mutation decreases LIMP-2 binding; d.a cell comprising a DNA sequence capable of expressing a GCase proteinor a peptide, wherein said peptide comprises an amino acid sequencecomprising from about 90% to about 100% homology to residues 347 through422 of SEQ ID NO 2, wherein said amino acid sequence binds to a LIMP-2receptor, and wherein said amino acid sequence is isolated orsynthesized; and e. or a combination thereof, to a patient in needthereof.
 37. The method of claim 36, wherein said disorder comprisesdefective GCase activity.
 38. The method of claim 37, wherein saiddefective activity comprises decreased enzymatic activity.
 39. Themethod of claim 36, wherein said disorder comprises alpha-synucleindysregulation.
 40. The method of claim 36, wherein said disorder is alysosomal storage disease.
 41. The method of claim 40, wherein saidlysosomal storage disease is selected from Gaucher disease, Fabrydisease, Pompe disease, mucopolysaccharidoses, and multiple systematrophy.
 42. The method of claim 36, wherein said disorder is aneurodegenerative disorder.
 43. The method of claim 42, wherein saidneurodegenerative disorder is selected from Parkinson disease, Alzheimerdisease, and Lewy body dementia.
 44. The method of claim 36, whereinsaid method comprises the step of administering said therapeutic agentvia intraventricular and/or stereotactic intracerebral injection. 45.The method of claim 36, wherein said dysfunction in the GCase pathwayresults in a central nervous system disease wherein the pathogenesis ofsaid disease results in plaques and/or disease states associated withplaques.